A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis.One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells.Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections.Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I.The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.
HIV-1 infections, if untreated, lead to acquired immunodeficiency syndrome (AIDS), which is marked by damaged immune system, increased chance of opportunistic infections, other severe illnesses, and death of the patients typically within 3 years.Although antiretroviral therapy (ART) has transformed the treatment of HIV infections and significantly reduced the death rate, currently available antiretrovirals cannot cure the infection.Latent viral reservoirs persist in the infected individuals and can lead to viral rebounds if ART is stopped (1).The life-long use of antiretrovirals, however, often leads to drug resistance and/or severe side effects (2).Novel antiretrovirals that can better treat, and ideally cure, HIV infections are therefore highly desired.
The HIV-1 accessory protein Nef is a small, multifunctional protein that plays an important role in viral pathogenesis (3).Nef expression in vivo is associated with high viral loads and disease progression into AIDS (4).Individuals infected by nefdefective HIV-1 strains do not develop AIDS for decades in the absence of ART (5)(6)(7)(8).This effect on viral pathogenesis presumably comes from Nef's ability to enable immune evasion.Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface, which disrupts antigen presentation and thus enables the infected cell to hide from the immune surveillance mediated by the CD8 + cytotoxic T lymphocytes (9,10).Nef also downregulates the CD4 receptor from the cell surface (11)(12)(13), which facilitates viral replication (14-17) and, importantly, allows the infected cell to evade antibody-dependent cellular cytotoxicity (18,19).The promise of Nef inhibition, therefore, is that it should revitalize these essential immune mechanisms to target and kill infected cells.Nef inhibition may be particularly relevant, or even necessary, in the shock and kill strategy (20).As proposed, the first step of this strategy is latency reversal, in which viral replication is reactivated therapeutically in infected cells (shock).Subsequently, host immunity would detect the cells that are undergoing active HIV-1 replication and clear them (kill).It is conceivable that, without inhibiting Nef, the "kill" step would be inefficient due to Nef-mediated immune evasion.
The benefits of Nef inhibition may also go beyond the scope of "kill"; it may facilitate latency reversal as well.It is known that Nef expression persists during ART (21,22).A recent study showed that, during the early stage of ART, the immune evasion activity of Nef-particularly the activity to cause MHC-I downregulation and thus evasion of cytotoxic T lymphocytes-correlates positively with viral reservoir size (23).Furthermore, in another study where observations were made after long-term ART, Nef-mediated immune evasion was found to protect genetically intact proviruses and thus contribute to HIV-1 persistence in effector memory CD4+ T cells (24).These findings suggest that successful inhibition of Nef, if achieved, should interfere with the establishment and/ or maintenance of latent reservoirs.
Despite the great promise, however, development of Nef inhibitors has been challenging (25).Two issues may have contributed to difficulties here.First, Nef lacks a defined pocket, and previous inhibitor development likely did not target the specific surface/pocket of Nef that is involved in Nef-mediated immune evasion.Second, previous efforts may have suffered from the conformational plasticity of Nef.As a master of protein-protein interactions, Nef is structurally versatile.Nef has a rigid core domain as well as two long flexible loops, one at the N-terminus and another close to the C-terminus.These flexible loops adopt different conformations when Nef interacts with different protein partners.
High-resolution structures solved by us have revealed the mechanistic details of Nef-mediated downregulation of MHC-I and CD4, respectively, which provided clues to overcome the above-mentioned challenges in Nef inhibitor development (26,27).First, a conserved pocket of Nef was found to be involved in both activities of Nef, suggesting that this pocket of Nef should be the focus for inhibitor development (27).Second, these structures revealed the specific conformation of Nef involved in each downregulation and, importantly, how each conformation is achieved and/or stabilized by Nef's association with the hijacked clathrin AP complex.
Nef downregulates MHC-I by intervening with the anterograde transport of newly synthesized MHC-I.At the trans-Golgi network, Nef orchestrates a three-molecule association between itself, the cytoplasmic domain of MHC-I (MHC-I CD ), and the clathrin adaptor protein 1 (AP1) (26,(28)(29)(30)(31)(32).Through this interaction, the peptide-loaded MHC-I, instead of being transported to the cell surface, is redirected toward the endolysosomal pathway and eventually degraded in the lysosome.As revealed by our crystal structure, Nef binds exclusively to the C-terminal domain of the m1 subunit (m1 CTD ) of AP1.An elongated "furrow" is then formed at the Nef-m1 CTD interface, and the MHC-I CD binds snugly into it (Fig. 1A) (26).This MHC-I CD -binding site is of a distinctive shape and may be targeted by inhibitors to disrupt Nef-mediated MHC-I downregulation.MHC-I CD -binding covers an interface area of 1208 Å 2 , which is greater than what is believed to be optimal for inhibition by small molecules (binding surface ≤ 1000 Å 2 ).However, given that the interaction between Nef, MHC-I, and m1 is of a highly cooperative, delicate nature ( 26), we believe that the complex formation here should be sensitive to small molecule inhibitors.).B, SDS PAGE analysis of purified MBP-Nef-m1 CTD .C, while Y320 of MHC-I binds to the tyrosine-binding pocket of m1, the m1 pocket designated for binding F is unoccupied because of the presence of a small Ala residue at position 323 of MHC-I.D, FP assays assessing the binding between the fluorescence probes and MBP-Nef-m1 CTD .Each data point is shown as the averaged FP from three independent samples ± SD.Each data series was fitted with a nonlinear regression curve using the one-site binding method.The TMRmutant-MHC-I CD curve is informative in two ways: at lower concentrations of MBP-Nef-m1 CTD , it indicates the baseline of the FP signal; at higher concentrations of MBP-Nef-m1 CTD , it shows the noise signal caused presumably by the light scattering of MBP-Nef-m1 CTD .FP signals generated using TMR-MHC-I CD as the probe are above the baseline but modestly so.FP signals generated using TMR-enhanced-MHC-I CD as the probe are sufficiently higher than the baseline.

FP assay for HTS against Nef-mediated MHC-I downregulation
Inspired and guided by these structure-derived insights, we have designed, developed, and optimized a fluorescence polarization (FP) assay for identifying such inhibitors through highthroughput screening (HTS).

Construct design and initial FP assay using TMR-MHC-I CD
We reasoned that an FP assay using a fluorescently labeled MHC-I CD , if developed successfully, should work competently in HTS to identify the desired inhibitors-compounds that can associate into the pocket at the Nef-m1 CTD interface and thus block the recruitment of MHC-I.We then followed the threestep protocol described by Moerke to develop such an FP assay (33).We first designed and commercially synthesized the fluorescent probe: a linear MHC-I CD peptide with a fluorescent tag, tetramethylrhodamine (TMR), attached at the Nterminus.We then designed and created a Nef-m1 CTD fusion construct capable of binding MHC-I CD and thus suitable for the FP assay.As described above, the tri-molecular association between Nef, MHC-I CD , and m1 CTD is cooperative in nature; in the absence of MHC-I CD , the binary association between Nef and m1 CTD is of low affinity (K D estimated to be in the micromolar range) and dissociation-prone (26).To ensure the formation of the binary complex between Nef and m1 CTD and thus that of the MHC-I-binding pocket, we fused Nef to the N-terminus of m1 CTD via a 20-amino-acid linker, which, according to the structure, should be flexible enough to not interfere with Nef's binding with m1 CTD .The Nef-m1 CTD fusion, carrying an N-terminal MBP tag, was expressed and purified to homogeneity in high yield (Fig. 1B).
For the FP assay, a working concentration of 50 nM was used for the TMR-MHC-I CD probe because the emitted fluorescence intensity at this concentration is more than 10 times greater than the background signal (buffer only).The assay volume was miniaturized and kept at 15 ml for compatibility with HTS.As expected, when the concentration of the Nef-m1 CTD fusion increased, the FP signal increased accordingly, which is consistent with the TMR-MHC-I CD probe binding to the large Nef-m1 CTD fusion protein leading to slower tumbling of the fluorophore and thus increased FP signal (Fig. 1C, red curve).In contrast, in the control experiment using a TMR-mutant-MHC-I CD probe, which carries both Y320D and D327R mutations and is thus incapable of binding to Nef-m1 CTD , the FP signal stayed mostly at the background level, although it increased modestly after the Nef-m1 CTD concentration increased beyond 50 mM (Fig. 1C, blue curve).Here, the abovebackground FP signal observed at concentrations of Nef-m1 CTD higher than 50 mM indicates that a significant amount of noise exists in this concentration range.We believe that the noise signal is a result of light scattering caused by high concentrations of the large MBP-Nef-m1 CTD fusion protein (Fig. 1C).

Using TMR-enhanced-MHC-I CD improves the competency of the FP assay
Although noise-free FP signal was successfully observed at low concentrations of Nef-m1 CTD in our initial tests (Fig. 1C, red curve), the signal window (difference between the red and the blue curves, Fig. 1C) was very modest.We reasoned that the low affinity binding of TMR-MHC-I CD to the Nef-m1 CTD fusion could be what is limiting the signal window.We therefore sought to design a new probe that could bind the Nef-m1 CTD fusion with higher affinity.
MHC-I contains a defective tyrosine-based sorting motif in its cytoplasmic tail.The canonical motif, denoted as YxxF, binds to the m subunits of different clathrin AP complexes.It contains, in addition to Tyr, a large, hydrophobic residue (F: Leu, Ile, or Met).MHC-I CD , however, lacks such a hydrophobic residue at this position and has a small Ala residue here instead.Thus, while the Tyr residue within MHC-I CD , Y320, binds to the canonical Tyr-binding pocket of m1, the m1 pocket designated for binding F is left unfilled because of the presence of an Ala residue at position 323 of MHC-I (Fig. 1D).Such a feature dictates that, in the absence of Nef, MHC-I does not bind to m1 efficiently and is therefore not trafficked through the AP1-dependent clathrin membrane trafficking pathway.This, however, offered an opportunity for our design of a new probe: converting the YxxA sequence within TMR-MHC-I CD into the canonical YxxF motif should help improve the binding affinity between the fluorescent probe and the Nef-m1 CTD fusion.We therefore created such a probe-replacing Ala323 with a Leu residue-and named it herein as TMRenhanced-MHC-I CD .Gratifyingly, the use of TMR-enhanced-MHC-I CD in the FP assay indeed resulted in higher-affinity binding: the K D improved from 44.1 mM to 18.8 mM when the probe switched from TMR-MHC-I CD to TMR-enhanced-MHC-I CD (Fig. 1C).More importantly, the TMR-enhanced-MHC-I CD probe helped improve the signal window of the assay in the region of low Nef-m1 CTD concentration (Fig. 1C).
The Nef-m1 CTD concentration of 30 mM was selected, which gave a signal window of more than 50 mP and should allow sufficient sensitivity to competition (here, the equilibrium concentrations of the probe-protein complex and the free probe are calculated to be 30.7 nM and 19.3 nM, respectively).

Assessment of the assay's tolerance toward DMSO
Since compounds in most small molecule libraries are dissolved in dimethylsulfoxide (DMSO), we tested our assay for its tolerance of DMSO.As shown in Figure 2, DMSO concentrations of 1.6% or lower led to minimal, if at all, decrease of the FP signal.At DMSO concentrations of 3.1% or 6.3%, the FP signal window decreased by 10% only.Overall, our FP assay can be considered as stable at up to 6.3% of DMSO.Notably, for library screening, the assay needs to be tolerant of 1% of DMSO.

The optimized assay is responsive to competitive binding of an inhibitor
We next investigated whether our assay is sensitive to competitive inhibition.Here, an unlabeled, modified MHC-I CD peptide was used as the competitor.The sequence of this competitor peptide is different from the authentic sequence of MHC-I CD in the following ways.First, the same A323L mutation was introduced so that the complete YxxF motif was installed, which should allow this unlabeled peptide to compete efficiently with the TMR-enhanced-MHC-I CD probe for binding.Second, to improve the solubility of the competitor peptide, we introduced three additional mutations in the MHC-I CD sequence: Q322E, G325S, and S326D.According to the previous structure, these mutations should not interfere with Nef-m1 CTD binding but should increase the peptide's hydrophilicity.Adding this unlabeled, MHC-I CD -mimetic peptide as the competitor indeed led to a dose-dependent decrease of the FP signal, consistent with the expected displacement of the TMR-enhanced-MHC-I CD probe by the competitor (Fig. 3).

The FP assay is robust and suitable for HTS
To assess whether our assay is competent for HTS, we calculated the Z 0 factor.For the positive control, 600 mM of the unlabeled, modified MHC-I CD peptide was used to ensure the complete displacement of the TMR-enhanced-MHC-I CD probe.From 60 positive controls and 60 negative controls (Nef-m1 CTD with TMR-enhanced-MHC-I CD ), the Z 0 factor was calculated to be 0.64, indicative of an excellent assay for HTS (Fig. 4A).
To further assess the assay's specificity and suitability for screening, we tested the following tool compounds/molecules in the assay: chloramphenicol (a secondary metabolite), maltose (a carbohydrate), WT-MHC-I CD (a peptide), and MBP-CD4 CD (a chimeric protein containing MBP and the cytoplasmic tail of CD4).MBP-CD4 CD should help validate the specificity of our assay.As mentioned earlier, although Nef downregulates CD4, the CD4-binding pocket should be properly formed only when Nef is in complex with clathrin AP2.In contrast, the MHC-I-binding pocket formed at the Nef-m1 interface is of a distinct shape, which should not allow CD4 binding.The WT-MHC-I CD peptide is less capable than the TMR-enhanced-MHC-I CD probe in binding the MBP-Nef-m1 CTD fusion; thus, addition of WT-MHC-I CD should not decrease the FP signal in any way efficient or at all.Furthermore, maltose binds into the active site of MBP but should not bind the Nef-m1 CTD portion of the fusion protein; this compound can therefore test how our assay would respond to binding occurring at a site other than the targeted MHC-I CDbinding pocket.As expected, addition of these molecules at 300 mM did not lead to any meaningful decrease of the FP signal (Fig. 4B).Importantly, the assay also did not suffer from any significant noise (Fig. 4B).Furthermore, the background of the assay showed no fluctuation at all when these molecules were added (not shown).Results here were highly reproducible when the experiment was performed on different plates and different days.Overall, the measured Z 0 factor and the tests with tool compounds/molecules together indicate that our assay is competent and suitable for HTS (33).

Discussion
We have developed a robust, HTS-compatible FP-based assay for screening small molecule inhibitors that directly target the MHC-I-binding pocket of the Nef-AP1 complex.Our work here was enabled and facilitated by the crystal structure reported by us previously (26).The highly cooperative nature of the binding, as revealed by the structure, prompted us to design a fusion protein to favor the formation of the MHC-I-binding "furrow" at the Nef-m1 interface (Fig. 1AB).In addition, the structural revelation that MHC-I binds to the conserved pocket on m1 using a "defective" YxxF motif inspired us to "restore" a canonical sorting motif in our MHC-I CD -based fluorescent probe, which was key in Right y axis: percentage of the FP signal correlated with binding (100% correlates with the FP signal of the optimized assay without DMSO added; 0% correlates with the background FP signal).Each data point is shown as the averaged FP of three independent samples ± SD. Analysis using oneway ANOVA indicates that this data is statistically significant (p < 0.0001).DMSO, dimethylsulfoxide.enabling our assay to achieve the desired signal window (Fig. 1CD).Our work here further illustrates how highresolution structures may inspire efforts toward developing drugs against challenging targets.
There are, however, some limitations with our assay here, which should be taken into account during the operation of the HTS.First, in the competition experiment, the FP signal was stable up to 4 h after addition of the competitor (Fig. 3); longer incubation led to a gradual decrease of the FP signal, although the dose-dependent trend was maintained (not shown).This time window is large enough for assay setup and plate reading but is nonetheless a limiting factor.To accommodate this, we may need to limit the number of libraries to be screened on a single day of operation.Second, according to our data (Fig. 3), high concentrations of the competitor were needed to displace the TMR-enhanced-MHC-I CD probe (IC 50 = 170 mM).We suspect that the effective concentration of our competitor could be much lower than the apparent concentration.We have noticed during our experiments that the competitor-unlabeled enhanced MHC-I CD peptidesometimes forms precipitates, indicating that it still has solubility issues.It is therefore possible that only a portion of this peptide was in the aggregation-free state and thus capable of competing with the TMR-enhanced-MHC-I CD probe for binding Nef-m1 CTD .If this is true, then the actual IC 50 would be smaller and may thus look more reasonable.Whether or not the measured IC 50 of unlabeled enhanced MHC-I CD peptide is an understatement of its true competency as a competitor, it remains safe to conclude that this optimized assay is capable of detecting competitive binding at the targeted pocket.However, if the measured IC 50 is indeed high and over 100 mM competitor is needed to induce a statistically significant drop of the FP signal, it could mean that low-affinity binders may be missed as hits during HTS using this assay.
Parallel to this work, we have also developed, as reported in the companion publication (34), an FP assay for screening small molecule inhibitors that could disrupt HIV-1 Nef-mediated CD4 downregulation.We will use the two assays in parallel in HTS, which should help us identify true inhibitors of Nef with unique specificities (active against one or both functions of Nef).

Fusion protein design, expression, and purification
The Nef-m1 CTD fusion was constructed by fusing HIV-1 Nef  FP assay for HTS against Nef-mediated MHC-I downregulation Triton X-100, pH 8.0).A stock protein solution of 120 mM Nef-m1 CTD was then prepared and was subsequently used to create different dilutions.Assays were carried out in Corning 384-well black microplates (3820).In each well, 50 nM TMRlabeled MHC-I CD peptide was mixed with MBP-Nef-m1 CTD at varied concentrations in a total volume of 15 ml.The plate was incubated for 12 h at room temperature with minimal exposure to light.FP was then measured using the EnVision plate reader (PerkinElmer) with excitation at 535 nm and emission at 595 nm.Experiments were done in triplicates.Data was fitted to nonlinear regression and plotted as a function of protein concentration in a logarithmic scale using OriginLab.
The probes used were synthesized by GenScript, including the following: TMR-MHC-I CD : TMR-SYSQAAGSDSAQ; TMR-enhanced-MHC-I CD : TMR-SYSQLAGSDSAQ; TMRmutant-MHC-I CD : TMR-SDSQAAGSRSAQ.These probes are stable at room temperature on the bench for at least 20 h.
For calculating the dissociation constants (K D ), the noise, represented by the FP signals generated with TMR-mutant-MHC-I CD being used as the probe, was first subtracted from the measured FP signals with TMR-MHC-I CD and TMRenhanced-MHC-I CD , respectively.The adjusted data was then fitted to nonlinear regression for one-site binding using the following equation: Y = B max *X/(K D + X) (B max : maximum specific binding in the same units as Y).

DMSO tolerance
Assay solutions were prepared containing 30 mM MBP-Nef-m1 CTD , 50 nM TMR-enhanced-MHC-I CD , and different concentrations of DMSO (0-25%).After incubation at room temperature for 2 h, FP values were measured and recorded.Experiments were done in triplicates.

Competition of TMR-enhanced-MHC-I by unlabeled enhanced-MHC-I
For competition using unlabeled enhanced-MHC-I CD peptide, a stock solution of 1.2 mM unlabeled enhanced-MHC-I CD peptide was first prepared.The stock solution was then serial-diluted (2-fold each) 10 times.For making the final assay solutions, MBP-Nef-m1 CTD and TMR-enhanced-MHC-I CD were first mixed and incubated at room temperature for 1 h.Then, unlabeled enhanced-MHC-I CD peptide was added (final concentrations: 30 mM MBP-Nef-m1 CTD , 50 nM of TMRenhanced-MHC-I CD , and varied concentrations of unlabeled enhanced-MHC-I CD peptide).The plate was incubated at room temperature and read at different time points.FP values were recorded.All experiments were done in triplicates.

Determination of the Z 0 factor
Negative control contains 30 mM of MBP-Nef-m1 CTD , 50 nM TMR-enhanced-MHC-I CD , and 2% DMSO.Positive control contains 30 mM MBP-Nef-m1 CTD , 50 nM TMRenhanced-CD4 CD , 600 mM unlabeled enhanced-MHC-I CD , and 2% DMSO.Samples of positive controls and negative controls (60 wells each) were prepared in a 384-well plate and incubated at room temperature for 2 h.FP values were recorded using the plate reader.The Z 0 factor was calculated using the following equation: Where m N and m P are the averages of mP values of negative and positive controls, respectively.SD N and SD P are the SDs.

Data analysis
Nonlinear regression fitting of the FP data was done using OriginLab.Statistical analysis was performed using Ordinary one-way ANOVA in GraphPad Prism.A p value of < 0.05 is considered statistically significant.

Figure 1 .
Figure 1.Structure-guided design and initial tests of the FP assay.A, a cooperative three-protein binding places MCH-I CD in a "furrow" formed at the interface between Nef and the C-terminal domain of the m1 subunit of AP1 (PDB ID: 4en2).B, SDS PAGE analysis of purified MBP-Nef-m1 CTD .C, while Y320 of MHC-I binds to the tyrosine-binding pocket of m1, the m1 pocket designated for binding F is unoccupied because of the presence of a small Ala residue at

Figure 2 .
Figure 2. Effect of DMSO on the FP assay.Left y axis: FP signal (mP) of the assay normalized by substracting out the background (buffer only) FP signal; the obtained DmP values here should correlate directly with binding.

Figure 3 .
Figure 3. Dose-dependent competition of the fluorescence-labeled probe by an unlabeled, MHC-I CD -mimetic peptide.Competition was successfully observed after 1-h incubation, and the signals remained stable after 4 h.No further time points were shown.Each data point is shown as the averaged FP of three independent samples ± SD. Analysis using oneway ANOVA indicates that the data is statistically significant (p < 0.0001).

(
59-206, NL4.3) to the C terminal domain of m1 subunit of AP1 via a flexible linker of 20 amino acids.Gene encoding the above Nef-m1 CTD fusion was cloned into a modified pMAT9 expression vector with a 6xHis tag introduced at the C-terminus of the fusion protein.Escherichia coli cells transformed with the plasmid were grown at 37 C till A 600 reached 0.8.Protein expression was then induced with 0.1 mM IPTG and continued at 16 C overnight.Cells were then lysed using sonication.The protein of interest was purified sequentially through a Ni-NTA affinity column, a MBP affinity column, a HiTrap Q anion exchange column, and finally a Superdex 200 size exclusion column.Fluorescence polarization assay using Nef-m1 CTD and the TMRenhanced-MHC-I CD peptide Purified MBP-Nef-m1 CTD was buffer exchanged into the assay buffer (50 mM Tris, 150 mM NaCl, 0.5 mM DTT, 0.01%

Figure 4 .
Figure 4. Assessing the assay's suitability for HTS.A, measurement of the Z 0 factor.The mean values of positive and negative controls are indicated by the dashed lines, while solid lines indicate the range where data points were considered.B, tool compounds did not cause noise in the assay.Chloramphenicol, maltose, WT-MHC-I CD peptide (319-330 of MHC-I), and MBP-CD4 CD (394-419) was each added to the assay solution to a final concentration of 300 mM (final dimethylsulfoxide concentration = 3%).Negative and positive controls were prepared the same way as in A.